Modelling human pathology of traumatic brain injury in animal models.

Traumatic brain injury (TBI) is caused by a head impact with a force exceeding regular exposure from normal body movement which the brain normally can accommodate. People affected include, but are not restricted to, sport athletes in American football, ice hockey, boxing as well as military personnel. Both single and repetitive exposures may affect the brain acutely and can lead to chronic neurodegenerative changes including chronic traumatic encephalopathy associated with the development of dementia. The changes in the brain following TBI include neuroinflammation, white matter lesions, and axonal damage as well as hyperphosphorylation and aggregation of tau protein. Even though the human brain gross anatomy is different from rodents implicating different energy transfer upon impact, especially rotational forces, animal models of TBI are important tools to investigate the changes that occur upon TBI at molecular and cellular levels. Importantly, such models may help to increase the knowledge of how the pathologies develop, including the spreading of tau pathologies, and how to diagnose the severity of the TBI in the clinic. In addition, animal models are helpful in the development of novel biomarkers and can also be used to test potential disease-modifying compounds in a preclinical setting.

Guidelines for conducting epidemiological studies of blast injury.

Blast injuries are often caused by more than one mechanism, do not occur in isolation, and typically elicit a secondary multi-system response. Research efforts often do not separate blast injuries caused by blast waves from those caused by blunt force trauma and other mechanisms. 15 experts from nine different NATO nations developed in the HFM Research Task Group (RTG; HFM-234 (RTG)) 'Environmental Toxicology of Blast Exposures: Injury Metrics, Modelling, Methods and Standards' Guidelines for Conducting Epidemiological Studies of Blast Injury. This paper describes these guidelines, which are intended to provide blast injury researchers and clinicians with a basic set of recommendations for blast injury epidemiological study design and data collection that need to be considered and described when conducting prospective longitudinal studies of blast injury.

Guidelines for using animal models in blast injury research.

Blast injury is a very complex phenomenon and frequently results in multiple injuries. One method to investigate the consequences of blast injuries is with the use of living systems (animal models). The use of animals allows the examination and evaluation of injury mechanisms in a more controlled manner, allowing variables such as primary or secondary blast injury for example, to be isolated and manipulated as required. To ensure a degree of standardisation across the blast research community a set of guidelines which helps researchers navigate challenges of modelling blast injuries in animals is required. This paper describes the guidelines for Using Animal Models in Blast Injury Research developed by the NATO Health Factors and Medicine (HFM) Research Task Group 234.

Mechanisms of blast induced brain injuries, experimental studies in rats.

Traumatic brain injuries (TBI) potentially induced by blast waves from detonations result in significant diagnostic problems. It may be assumed that several mechanisms contribute to the injury. This study is an attempt to characterize the presumed components of the blast induced TBI. Our experimental models include a blast tube in which an anesthetized rat can be exposed to controlled detonations of explosives that result in a pressure wave with a magnitude between 130 and 260 kPa. In this model, the animal is fixed with a metal net to avoid head acceleration forces. The second model is a controlled penetration of a 2mm thick needle. In the third model the animal is subjected to a high-speed sagittal rotation angular acceleration. Immunohistochemical labeling for amyloid precursor protein revealed signs of diffuse axonal injury (DAI) in the penetration and rotation models. Signs of punctuate inflammation were observed after focal and rotation injury. Exposure in the blast tube did not induce DAI or detectable cell death, but functional changes. Affymetrix Gene arrays showed changes in the expression in a large number of gene families including cell death, inflammation and neurotransmitters in the hippocampus after both acceleration and penetration injuries. Exposure to the primary blast wave induced limited shifts in gene expression in the hippocampus. The most interesting findings were a downregulation of genes involved in neurogenesis and synaptic transmission. These experiments indicate that rotational acceleration may be a critical factor for DAI and other acute changes after blast TBI. The further exploration of the mechanisms of blast TBI will have to include a search for long-term effects.

Cyanoacrylate in nerve repair: transient cytotoxic effect.

Cyanoacrylate adhesive has been suggested as an alternative to suturing when repairing severed peripheral nerves. The authors examined the cytotoxic effect of ethyl-cyanoacrylate on the human neuroblastoma cell line SH-SY5Y and compared it with the effects of butyl-cyanoacrylate (Histoacryl), an adhesive approved for skin closure. Ethyl-cyanoacrylate or butyl-cyanoacrylate was applied in confluent SH-SY5Y cultures. Immediately, at 24h and at 7, 14, 21 and 28 days, cultures were photographed and analysed digitally. At corresponding intervals, cell death was quantified using a (51)Cr release assay. In cultures exposed to ethyl-cyanoacrylate or butyl-cyanoacrylate, cell death was observed predominantly in conjunction with the adhesive, causing a halo devoid of cells. Surviving cells showed neurodegenerative properties with loss of neuritis and reduction of body size up to 3 days post exposure. The inhibition halo diminished over time in both groups and at 28 days cells reached the margin of the adhesive in the ethyl-cyanoacrylate group. (51)Cr assay indicated significant cell death in exposed cultures, which rapidly decreased during the first 14 days. No significant differences were found between the adhesives. This study demonstrates that ethyl-cyanoacrylate and butyl-cyanoacrylate have a transient cytotoxic effect, which may explain the promising results when using cyanoacrylate for nerve repair.

Differential gene expression in the rat cochlea after exposure to impulse noise.

Understanding the molecular biology of noise trauma is vital to developing effective and timely interventions. In a model of explosion-mediated impulse noise injury, differential gene expression was studied in whole rat cochlea preparations at 3 and 24 h following the exposure. We developed a technique using mRNA from a single cochlea on each oligonucleotide microarray to avoid pooling of mRNA samples. Application of a conservative statistical analysis approach resulted in the identification of 61 differentially expressed genes. Within 3 h after the exposure, there was an up-regulation of immediate early genes, mainly transcription factors and genes involved in the tissue's response to oxidative stress. No genes were found to be significantly down-regulated. At 24 h following the exposure, up-regulated genes included members of inflammatory and antioxidant pathways and one gene involved in glutathione metabolism was down-regulated. A subset of genes was confirmed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The present study demonstrates the power of the microarray technique in providing a global view of the gene regulation following noise exposure, and in identifying genes that may be mechanistically important in hearing loss, and thereby serve as a basis for the development of therapeutic interventions.

Semaphorin and neuropilin expression in motoneurons after intraspinal motoneuron axotomy.

We have examined mRNA and protein distribution for the axon guidance molecules semaphorin3A, 3F, 4F and semaphorin receptors neuropilin-1 and 2, 1-21 days after intramedullary axotomy of rat lumbar spinal cord motoneurons. We show that semaphorin3A mRNA and protein are up-regulated in the scar and in motoneurons from 3 days and upto 3 weeks after injury. Neuropilin-1 mRNA showed no changed expression in axotomized motoneurons. Semaphorin3F mRNA expression was found in ventral roots after ventral funiculus lesion (VFL) and neuropilin-2 mRNA was found in affected motoneurons from 1 day after injury throughout the examined period. Semaphorin4F mRNA was first found in motoneurons 3 weeks after lesion. These results suggest semaphorin/neuropilin involvement in the injury response of intramedullary axotomized motoneurons.

Expression of ErbB3, ErbB4, and neuregulin-1 mRNA during tooth development.

The receptor tyrosine kinases ErbB3 and ErbB4, which bind to various variants of neuregulin (NRG), play fundamental roles in neural development and in organs, which form through epithelial-mesenchymal interactions. Here, we demonstrate that NRG-1 and the receptors ErbB3 and ErbB4 are expressed locally during rodent tooth development. However, the mRNA expression patterns of ErbB3 and ErbB4 were distinctly different during odontogenesis. Examinations of teeth in genetically heart-rescued ErbB4-/- mice did not reveal any obvious deviation from the normal phenotype. The results suggest that ErbB3 and ErbB4 may participate in tooth morphogenesis. The specific interactions between NRG isoforms and ErbB receptors during this process remain to be determined.

Expression of neuregulin and ErbB3 and ErbB4 after a traumatic lesion in the ventral funiculus of the spinal cord and in the intact primary olfactory system.

Neuron-derived neuregulins have been implicated in the regulation of glial cell function and survival. This factor family and its receptors may therefore be assumed to be of importance for the cellular response to traumatic injury. In this study we have examined the distribution of mRNA for neuregulin 1 (NRG1), ErbB3 and ErbB4-receptor tyrosine kinases after a ventral funiculus lesion in the lumbar spinal cord (VFL). The techniques used were in situ hybridization and immunohistochemistry. The survival times were 1-21 days. The spinal cords from normal adult and embryonic rats were used as controls. For comparison, sections from the olfactory bulb of perinatal and adult rats were also included in the study. Expression of NRG1 mRNA was observed in motoneurons in the intact spinal cord. A decrease in the labeling for NRG1 mRNA was seen during the first 5 days after VFL but then became slightly upregulated at 3 weeks after the lesion. A high labeling signal for ErbB3-mRNA was observed in the ventral and dorsal roots of E16 and E18 embryos. Labeling for ErbB3-mRNA was strong in the affected ventral root at 3 days after the VFL, reached a maximum at 1 week and was still upregulated after 3 weeks. Increased labeling for ErbB3 was also noted in scattered cells in the scar tissue 1-3 weeks after the VFL. These findings were verified with immunohistochemistry for ErbB3. A strong labeling for ErbB3 in the olfactory nerve fiber layer and olfactory nerve bundles was observed in rats of all ages examined. ErbB4 had strong expression in the embryonic spinal cord, but no evidence for lesion-induced regulation of ErbB4 receptors could be found after the VFL. Our data show that ErbB3 in the ventral roots was upregulated after a VFL and that NRG1 mRNA was initially downregulated in the motoneurons. The lesion-induced changes in the expression of NRG1 and ErbB3 in the injured spinal cord and denervated ventral root can be assumed to be of importance for axonal growth and the regulation of glial cell survival.

Properties of motoneurons underlying their regenerative capacity after axon lesions in the ventral funiculus or at the surface of the spinal cord.

Spinal motoneurons represent neurons with axons located in both the central (CNS) and peripheral (PNS) nervous systems. Following a lesion to their axons in the PNS, motoneurons are able to regenerate. The regenerative capacity of these neurons is seen also after lesion in the ventral funiculus of the spinal cord, i.e. within the CNS compartment. Thus, after an axotomy within the ventral funiculus, motoneurons respond with a changing polarity towards production of axons, sometimes even from the dendritic tree. This capacity can be used in cases of ventral root avulsion (VRA) lesions, if a conduit for outgrowing axons is presented in the form of replanted ventral roots. In human cases, this procedure may accomplish return of function in denervated muscles. The strong regenerative capacity of motoneurons provides the basis for studies of the response in motoneurons with regard to their contents of substances related to survival and regeneration. Such studies have shown that, of the large number of receptors for neurotrophic substances and extracellular matrix molecules, mRNAs for receptors or receptor components for neurotrophin-3 (NT-3), ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) are strongly downregulated after VRA, while receptors for glial cell line-derived neurotrophic factor (GDNF) and laminins are profoundly upregulated. These results should be considered in the design of combined pharmacological and surgical approaches to lesions of motor axons at or close to the CNS-PNS interface.

Expression of tenascin R and J1 mRNA in motoneurons after a traumatic lesion in the spinal cord.

We demonstrate, using in situ hybridization, that mRNA for the anti-adhesive molecules tenascin R and J1 in the adult rat spinal motoneurons are down-regulated rapidly as a reaction after a ventral funiculus lesion. Tenascin-R was significantly down-regulated at day 1 and normalized after 3 weeks. Tenascin-J1 declined to its lowest value at day 3 and returned to the initial level after 3 weeks. In adjacent sections, the distribution of macrophages was studied with immuno histochemistry. The density of macrophages reached a maximum 3 days after the injury. Thus, the density of macrophages appeared to be inversely related to the level of tenascin mRNA. These data are compatible with the notion that neuronal tenascins may modulate the adhesion of perincurial inflammatory cells.

Distribution of TGF-beta, the TGF-beta type I receptor and the R-II receptor in peripheral nerves and mechanoreceptors; observations on changes after traumatic injury.

The mechanisms governing the regeneration of denervated peripheral mechanoreceptors are similar to those of peripheral nerves. The ability to regenerate depends partly on changes of the Schwann cell phenotype. The transforming growth factor beta (TGF-beta) family have been implicated in induction of Schwann cell proliferation, production of extracellular matrix and neurotrophin synthesis as well as synthesis or repression of cell adhesion molecules. Hence, they may prove to be of importance for regenerative mechanisms in peripheral mechanoreceptors. The distribution of TGF-beta, the receptors I and II and intra-cellular second messengers, Smad 2/3 and 4 was assessed in sensory neurones, peripheral nerves and mechanoreceptors by immuno-histochemistry, immuno-electron microscopy and in situ hybridisation. TGF-beta2 mRNA and TGF-beta2-like immunoreactivity (IR) were expressed in injured small and medium sized rat sensory neurones of dorsal root ganglia. TGF-beta and receptor II mRNA and immunoreactivities (IR) were present in satellite cells. Intact and injured sensory neurones expressed receptor I mRNA and Smad 2 mRNA. TGF-beta2 mRNA was found in transected nerve stumps and in sensory mechanoreceptors. TGF-beta1, 2 and Smad 4 were also observed in inner core lamellar cells of intact and denervated cat Pacinian corpuscles. Lamellar cells of intact and denervated Meissner corpuscles were TGF-beta immunoreactive. Merkel cells were receptors I and II immunoreactive. In conclusion, cutaneous and subcutaneous mechanoreceptors differ with regard to the expression of TGF-beta isoforms and receptors.

Distribution of the neurotrophin receptors p75 and trkB in peripheral mechanoreceptors; observations on changes after injury.

The neurotrophin family mediates effects of growth, cell differentiation and cell death through low- and high-affinity transmembrane receptors. The Pacinian corpuscle (PC) is the largest peripheral mechanoreceptor in mammals and was studied by immuno-histochemistry and immuno-electron microscopy with regard to the distribution of neurotrophin receptors, p75; p140 trkA, p145 trkB and 145 trkC. TrkA- and trkC-like immunoreactivity (IR) was not expressed in rat and cat PCs. Developing and adult animals expressed p75 and trkB in lamellar cells of the PC. The inner core cells, thought to be specialised Schwann cells, demonstrated an injury-induced increased immuno-labelling for trk B. Perineurial-derived outer core cells were reactive to p75 after injury similar to the perineurium of distal nerve stumps. Inner core cells of PCs behaved as leptomeningeal cells with regard to trkB. Outer core lamellar cells of PCs behaved as perineurial cells with regard to p75. A role for brain-derived neurotrophic factor is proposed in the development and nerve regeneration of PCs via an anterograde messenger transfer through p75 and trkB.

Regulation of laminin-associated integrin subunit mRNAs in rat spinal motoneurons during postnatal development and after axonal injury.

Two important prerequisites for successful axon regeneration are that appropriate extracellular molecules are available for outgrowing axons and that receptors for such molecules are found in the regenerating neuron. Laminins and their receptors in the integrin family are examples of such molecules, and laminin-associated integrin subunits alpha 3, alpha 6, alpha 7, and beta 1 mRNAs have all been detected in adult rat motoneurons. We have here, by use of in situ hybridization histochemistry, examined the normal postnatal development of the expression in motoneurons of these mRNAs and integrin beta 4 mRNA, all of which have been associated with laminin-2. We studied the regulation of these mRNAs, 1-42 days after two types of axotomy in the adult rat (sciatic nerve transection, SNT; ventral root avulsion, VRA) and 1-10 days after SNT in the neonatal animal. During postnatal development, there was a distinct shift in the integrin composition from a stronger expression of the alpha 6 subunit to a very clear dominance of alpha 7 in the adult. All types of axotomy in the adult rat induced initial (1-7 days) large up-regulations of alpha 6, alpha 7 and beta1 subunit mRNAs (250-500%). Only minor changes for alpha 3 mRNA were seen, and beta 4 mRNA could not be detected at all in motoneurons. After adult SNT, the alpha 7 and beta 1 subunits were up-regulated throughout the studied period, and the alpha 6 subunit mRNA was eventually normalized. After VRA, however, the alpha 7 and beta1 levels peaked earlier than after SNT and were normalized at 42 days, whereas alpha 6 mRNA was up-regulated longer than after SNT. Neonatal SNT had much smaller effects on the expression of the studied subunits. The results suggest that an important part of the response to axotomy of motoneurons is to up-regulate receptors for laminin. The developmental shift in integrin subunit composition and the various responses seen in the lesion models indicate that different isoforms of laminin play a role in the regenerative response.

Differential expression of tenascin-C, tenascin-R, tenascin/J1, and tenascin-X in spinal cord scar tissue and in the olfactory system.

The members of the tenascin family are involved in a number of developmental processes, mainly by their ability to regulate cell adhesion. We have here studied the distribution of mRNAs for tenascin-X, -C, and -R and the closely related molecule tenascin/J1 in the olfactory system and spinal cord. The olfactory bulb and nasal mucosa were studied during late embryonic and early postnatal development as well as in the adult. The spinal cord was studied during late embryonic development and after mechanical lesions. In the normal rat, the spinal cord and olfactory bulb displayed similar patterns of tenascin expression. Tenascin-C, tenascin-R, and tenascin/J1 were all expressed in the olfactory bulb and spinal cord during development, while tenascin/J1 was the only extensively expressed tenascin molecule in the adult. In both regions tenascin/J1 was expressed in both nonneuronal and neuronal cells. After a spinal cord lesion, mRNAs for tenascin-C, -X, -R, and/J1 were all upregulated and had their own specific spatial and temporal expression patterns. Thus, even if axonal outgrowth occurs to some extent both in the adult rat primary olfactory system and in spinal cord scar tissue after lesion, the tenascin expression patterns in these two situations are totally different.

Differential regulation of trophic factor receptor mRNAs in spinal motoneurons after sciatic nerve transection and ventral root avulsion in the rat.

After sciatic nerve lesion in the adult rat, motoneurons survive and regenerate, whereas the same lesion in the neonatal animal or an avulsion of ventral roots from the spinal cord in adults induces extensive cell death among lesioned motoneurons with limited or no axon regeneration. A number of substances with neurotrophic effects have been shown to increase survival of motoneurons in vivo and in vitro. Here we have used semiquantitative in situ hybridization histochemistry to detect the regulation in motoneurons of mRNAs for receptors to ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) 1-42 days after the described three types of axon injury. After all types of injury, the mRNAs for GDNF receptors (GFRalpha-1 and c-RET) and the LIF receptor LIFR were distinctly (up to 300%) up-regulated in motoneurons. The CNTF receptor CNTFRalpha mRNA displayed only small changes, whereas the mRNA for membrane glycoprotein 130 (gp130), which is a critical receptor component for LIF and CNTF transduction, was profoundly down-regulated in motoneurons after ventral root avulsion. The BDNF full-length receptor trkB mRNA was up-regulated acutely after adult sciatic nerve lesion, whereas after ventral root avulsion trkB was down-regulated. The NT-3 receptor trkC mRNA was strongly down-regulated after ventral root avulsion. The results demonstrate that removal of peripheral nerve tissue from proximally lesioned motor axons induces profound down-regulations of mRNAs for critical components of receptors for CNTF, LIF, and NT-3 in affected motoneurons, but GDNF receptor mRNAs are up-regulated in the same situation. These results should be considered in relation to the extensive cell death among motoneurons after ventral root avulsion and should also be important for the design of therapeutical approaches in cases of motoneuron death.

Induction of VEGF and VEGF receptors in the spinal cord after mechanical spinal injury and prostaglandin administration.

Vascular endothelial growth factor (VEGF) is an angiogenetic factor that promotes endothelial cell proliferation during development and after injury to various types of tissue, including the central nervous system (CNS). Using immunohistochemical and in situ hybridization methods we have here demonstrated that VEGF and its receptors Flk-1, Flt-1 and Neuropilin-1 mRNAs and proteins are induced after incisions in the rat spinal cord. The inducible enzyme for prostaglandin synthesis cyclooxygenase-2 (COX-2) is known to be upregulated after spinal injury, cerebral ischemia and to stimulate angiogenesis. To test the hypothesis that prostaglandins may be involved in the VEGF response after lesion we investigated whether intraspinal microinjections of prostaglandin F2alpha (PGF2alpha) alters VEGF expression in the spinal cord. Such treatment was followed by a strong upregulation of VEGF mRNA and protein in the injection area. Finally, by use of an in vitro model with cell cultures of meningeal fibroblast and astrocyte origin, resembling the lesion area cellular content after spinal cord injury but devoid of inflammatory cells, we showed that VEGF is expressed in this in vitro model cell system after treatment with PGF2alpha and prostaglandin E2 (PGE2). These data suggest that cells within a lesion area in the spinal cord are capable of expressing VEGF and its receptors in response to mechanical injury and that prostaglandins may induce VEGF expression in such cells, even in the absence of inflammatory cells.

Ultrastructural evidence for a preferential elimination of glutamate-immunoreactive synaptic terminals from spinal motoneurons after intramedullary axotomy.

After axotomy in the ventral funiculus of the cat spinal cord, about half of the population of lesioned motoneurons die at 1-3 weeks postoperatively, whereas the other half survives and generates new axons through the lesion area. To identify conditions that may promote survival and regeneration of motoneurons subjected to this kind of injury, the authors examined ultrastructurally lesion-induced changes in the number and distribution of nerve terminals on the somata and proximal dendrites of alpha-motoneurons in the 7th lumbar spinal segment (L7) of the cat spinal cord. Intramedullary axotomy resulted in a profound reduction in the number of nerve terminals impinging on the somata and proximal dendrites, with the maximal effect seen at 3 weeks postlesion. At that time, only 12-25% of the normal number of terminals remained on the cell somata, and 22-33% remained on proximal dendrites. Thereafter, a gradual increase in terminal numbers occurred, reaching normal levels at 34 weeks after the lesion. Already at 2 days postoperatively and, most obviously, at 3 weeks postoperatively, type S nerve terminals were eliminated to a larger degree than type F terminals. Postembedding immunohistochemistry confirmed that the largest reduction at 3 weeks was seen for excitatory glutamate-immunopositive type S nerve terminals (90%), whereas inhibitory glycine-immunoreactive and gamma-aminobutyric acid (GABA)-immunoreactive type F terminals were affected less (70% reduction). This led to a distinct shift in the ratio between the numbers of terminals that were immunopositive for glycine and GABA and the numbers of terminals that were labeled for glutamate. For the cell body, this ratio increased from 3.7 in normal material to 14.5 in lesioned motoneurons, whereas the corresponding values for proximal dendrites were 3.8 and 7.5. The preferential elimination of glutamatergic inputs to lesioned motoneurons may reflect an active reorganization of the synaptic input to diminish the excitotoxic influence on these neurons, thereby promoting the survival of motoneurons after intramedullary axotomy.